Generation, characterisation and application of SARS-CoV-2 protein antigens for COVID-19 rapid diagnostic purposes in the hospital and community

我们的目标是产生一系列稳定转染的细胞系，以帮助满足对质量验证的重组SARS-CoV-2结构蛋白的需求，这些蛋白是迫切需要的，我们将使用它们来帮助开发诊断和疫苗。因此，我们的项目将补充和扩大COVID-19蛋白质门户网站上可用的蛋白质库。我们建立的平台也将为未来快速生成其他目标提供完美的定位。我们将在两种不同的哺乳动物表达系统（CHO和HEK293细胞）中生产这些细胞，通过质谱法对它们进行表征，并对Spike糖蛋白进行糖基化分析。在生产该蛋白的非糖基化形式进行比较时，我们将确定这种翻译后修饰对折叠、三聚体形式组装的重要性，以及该蛋白检测来自康复的COVID-19患者的抗刺突抗体的能力。SARS-CoV-2蛋白的重组真实和特征形式将在与我们的工业合作伙伴Mologic开发的诊断分析中进行评估，并在阿什福德的东肯特医院信托基金会进行ELISA分析，以分析康复的COVID-19患者的抗体反应。拟议的研究将解决世卫组织“2019年新型冠状病毒全球研究与创新协调全球研究路线图论坛”的问题，该论坛规定需要支持开发“可靠的血清学检测和监测反应的分析，并中期支持开发诊断产品，以改善临床流程，促进控制/护理点”和开发疫苗的试剂。

We aim to generate a range of stably transfected cell lines to help meet the demand for quality-verified recombinant SARS-CoV-2 structural proteins, which are desperately needed, and which we will use, to help in the development of diagnostics and vaccines. In so-doing our project will complement and expand the repertoire of proteins available on the COVID-19 Protein Portal. The platforms we establish will also be perfectly positioned for the rapid generation of other targets in the future. We will produce these in two different mammalian expression systems, CHO and HEK293 cells, characterise these by mass spectrometry and undertake glycosylation analysis of the Spike glycoprotein. In producing non-glycosylated forms of the protein for comparison, we will define the importance of this post-translational modification for folding, assembly of the trimer form and the ability of the protein to detect anti-spike antibodies from recovered COVID-19 patients. The recombinant authentic and characterised forms of the SARS-CoV-2 proteins will be assessed in diagnostic assays in development with our industrial partner Mologic, and in ELISA assays in the East Kent Hospital Trust at Ashford to profile antibody responses across recovered COVID-19 patients. The proposed research will address the WHO 'coordinated global research roadmap 2019 Novel Coronavirus Global Research and Innovation forum' that stipulates the need for reagents that support the development of 'reliable serological testing as well as assays that monitor response and midterm support development of diagnostic products to improve clinical processes to contribute to control/point of care' and for development of vaccines.